Random access microscopy (RAMP) relies upon positioning a point of illumination rapidly over a specimen with a programmable beam steering arrangement. Light emitted by the specimen is then detected with a camera or other detection source. For Super-RAMP microscopy, points of sparse illumination are scanned rapidly over the specimen. Subsequently, a sequence of images from the camera can be used to produce high-resolution, optically sectioned images.
One beam steering arrangement used in such techniques comprises one or more acousto-optic deflectors (AODs) operable to vary the deflection angle of a beam in response to variation in an applied acoustic frequency. In a typical arrangement, a pair of perpendicularly aligned AODs are used to separately control deflection along two perpendicular axes. AODs can be used to address any point in a field of view at random and, by limiting the number of points addressed, very high speeds of acquisition can be achieved. AODs have not been widely adopted for use in multi-photon microscopy because they cause significant temporal and spatial dispersion of the femtosecond pulses required for multi-photon activation. These effects reduce their efficiency and resolution unless they are properly compensated [12-18].
One compensation solution is to use an acousto-optic modulator (AOM) as a source of both spatial and temporal compensation [19]. If the AOM is positioned at 45 degrees and opposite to the direction of the perpendicularly aligned AODs, and the acoustic frequency applied to the AOM is carefully selected to match that applied to the AOD pair, this provides good compensation over a range of wavelengths [20]. The AOM also introduces negative group velocity displacement (GVD) and the distance between the AOM and AODs can be adjusted to provide optimal temporal compensation at different wavelengths [20].
Nevertheless, even using these techniques, there is still a limit to the displacement achievable using AODs and in particular to the displacement achievable without adversely impacting on the spatial profile of the beam. Typically, as the displacement increases, the beam profile becomes more elliptical. One way to combat the beam profile problem is to use large aperture AOMs and AODs but relatively small increases in the aperture size of such devices correlate to relatively large increases in their expense. Additionally, the illumination intensity also varies across the available scan area. The variation in illumination intensity is at least partially attributable to the substantially Gaussian profile of the beam upon entry to the AOM/AOD arrangement. These effects can reduce the efficiency and resolution achievable.
It is therefore an object of the present invention to provide an improved beam steering arrangement that at least partially overcomes or alleviates the above problems.